1. Field of the Invention
The present invention relates to methods for concentrating and recovering bacteria and free bacterial nucleic acids from biological samples. In particular, the present invention relates to methods for concentrating Mycobacterium tuberculosis in a manner that is compatible with subsequent nucleic acid analysis.
2. Background Information
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, is conventionally identified by time-consuming microbiological culture. Clinical specimens submitted for mycobacterial culture are often contaminated with other more rapidly growing microorganisms. These specimens, typically a sputum or other respiratory sample, must be subjected to a digestion-decontamination process to liquefy viscous organic material and eliminate unwanted organisms. The most common reagents used in the digestion-decontamination process are N-acetyl-L-cysteine-sodium hydroxide (NALC--NaOH), sodium hydroxide-sodium dodecyl sulfate (NaOH--SDS) or NaOH alone. A typical digestion-decontamination protocol would include incubating the respiratory sample with one of the above reagents, lowering the pH of the mixture by diluting with buffer solution or water, and centrifuging the mixture to concentrate the mycobacteria in a pellet. A portion of the supernatant would be decanted and the pellet resuspended in the remaining supernatant fluid or in a buffer solution. Suspensions obtained in such a manner are termed "respiratory sediments".
While respiratory sediments are suitable for culture, they are not suitable for nucleic acid analysis, such as nucleic acid amplification, restriction digestion, and nucleotide sequencing. Prior to conducting nucleic acid analysis, it is necessary to remove the digestion-decontamination reagents from the respiratory sediments because these reagents interfere with nucleic acid analysis. It is also useful to further concentrate the mycobacteria from sediment samples having low mycobacteria titers prior to nucleic acid analysis to increase the likelihood of nucleic acid detection.
Prior to the present invention, those skilled in the art attempted to solve the problems of contaminating digestion-decontamination reagents and low bacterial concentrations by diluting the samples in aqueous solutions, further centrifuging to pellet the mycobacteria, and discarding the supernatant solution containing the digestion-decontamination reagents. For example, Beavis et al. (J. Clin. Microbiol., 33, 2582-2586 (1995)) use a method in which 100 .mu.L of respiratory sediment is mixed with 500 .mu.L of a specimen wash reagent comprising Tris-HCl and 1% solubilizer. The mycobacteria are pelleted from the mixture by centrifugation at 12,500.times.g for 10 minutes. (See also, Roche Molecular Systems. 1994. Roche Amplicor Mycobacterium tuberculosis test insert, Roche Molecular Systems, Branchburg, N.J.) Such methods, however, have limitations. These methods are not particularly suitable for concentrating bacteria from large sample volumes because addition of the wash buffer increases the volume five-fold. In addition, such methods are limited because mycobacteria are very buoyant and are difficult to pellet from aqueous media. Thus, centrifugation in water or aqueous buffer solution, such as that utilized by Beavis et al., can result in significant loss of the target organisms. In addition, free mycobacterial nucleic acids in the respiratory sediment are not pelleted during centrifugation in aqueous medium. Free nucleic acids exist in the respiratory sediment because they can be released during bacterial lysis caused by digestion and decontamination of fresh samples, and by freezing and thawing of stored respiratory sediments.
The present invention overcomes these problems by providing a method for concentrating bacteria, including M. tuberculosis, from viscous biological samples. The present invention also provides a method for concentrating free bacterial nucleic acids as well as the bacteria present in biological samples. Samples processed according to the methods of the present invention can be used for subsequent nucleic acid analysis.